Controlled release reagent storage box for centrifugal flow control and micro-fluidic chip

Abstract

The invention discloses a controlled release reagent storage box for centrifugal flow control and a micro-fluidic chip, and belongs to the technical field of centrifugal micro-fluidic detection. The controlled-release reagent storage box comprises a box body and a sealing film covering the box body, a plurality of mutually independent reagent storage cavities are formed in the top of the kit body in a downward sunken mode, a release hole is formed in the position, close to the bottom wall, of the side wall of each reagent storage cavity, and a valve used for controlling reagent release is arranged in each release hole; one side of the box body extends outwards to form a convex block, and a sample adding opening is formed in the convex block. The micro-fluidic chip comprises a chip main body and a controlled release reagent storage box, the chip main body is provided with a containing cavity matched with the controlled release reagent storage box, and the controlled release reagent storage box is arranged in the containing cavity. Various reagents can be pre-stored, each reagent can be independently released, the sealing performance is good, and the stability of the reagents in the long-term storage process and the flexible release of the reagents in the reaction process are effectively ensured.

Description

Technical Field

[0001] The invention relates to the technical field of centrifugal microfluidic detection, and in particular to a controlled-release reagent storage box for centrifugal fluidics and a microfluidic chip. Background Art

[0002] With the rapid development of microfluidic technology, microfluidic platforms have been widely used in biomedicine, environmental monitoring, chemical analysis and other fields. Especially in the analysis of biological molecules such as nucleic acid extraction, purification and amplification, the portability, automation and integration advantages of microfluidic platforms are gradually emerging. Centrifugal microfluidics uses the centrifugal force generated by rotation to generate a pressure difference to drive the liquid to move in the microchannel, thereby achieving control of the flow, mixing, separation, reaction and other processes of the liquid. The above processes are all carried out in the microfluidic chip, so integrating the pre-storage and timed and quantitative release functions of multiple reagents in the microfluidic chip has become a key factor in promoting the integration and automation of microfluidic technology. It is usually necessary to pre-store a variety of reagents on a centrifugal microfluidic chip to perform multi-step reactions such as nucleic acid extraction, purification, and amplification. The current existing technology usually manually pre-adds a variety of reagents directly into the reagent chamber of the chip, and usually relies on different rotation speeds to release the reagents into the microchannel when in use. However, this method is unstable and complex in structure, and does not meet the characteristics of disposable, low-cost microfluidic chips. It is also impossible to independently release multiple reagents at different times in steps and in sequence, and it is difficult to meet the flexibility requirements of complex experimental processes with multiple steps and multiple reagents, resulting in the simplification of sample processing of microfluidic chips, the inability to integrate more reaction processes, and the difficulty in ensuring product recovery and purity due to human factors. At the same time, the method of directly pre-storing a variety of reagents in the chip is prone to leakage during long-term storage, which reduces the stability and sealing of the chip to a certain extent, thereby affecting the long-term storage effect and even the experimental results. Chinese patent CN CN110371435A provides a liquid reagent storage box that can be used in conjunction with a microfluidic chip, which uses heat to melt the sealing plug to open the storage cavity, thereby releasing the reagent. However, the reagent storage box of this structure can only store a single reagent and cannot store multiple reagents, so it cannot meet the needs of complex multi-step experimental processes.

[0003] Based on this, there is an urgent need to develop a controlled-release reagent storage box and a microfluidic chip that can achieve timed and stable release of multiple reagents after long-term pre-storage and is suitable for microfluidic chips. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a controlled-release reagent storage box for centrifugal fluidics, which can store a variety of reagents, and the release of each reagent can be independently controlled, with good sealing, effectively ensuring the stability of the reagent during long-term storage and the flexible release during the reaction process.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows: A controlled-release reagent storage box for centrifugal fluidics comprises a box body and a sealing film covering the box body; the top of the box body is recessed downward to form a plurality of mutually independent reagent storage chambers, a release hole is provided on the side wall of each reagent storage chamber near the bottom wall, a valve for controlling the release of the reagent is provided in the release hole; a convex block is extended outwardly from one side of the box body, and a sample addition port is provided on the convex block.

[0006] As a preferred embodiment of the present invention, the multiple reagent storage chambers are respectively a first cleaning liquid chamber, an elution liquid chamber, a second cleaning liquid chamber and a dilution liquid chamber; the elution liquid chamber is arranged between the first cleaning liquid chamber and the second cleaning liquid chamber, and the second cleaning liquid chamber is arranged between the elution liquid chamber and the dilution liquid chamber.

[0007] As a preferred embodiment of the present invention, an avoidance groove is provided on the bottom surface of the box body at a position corresponding to between the second cleaning liquid chamber and the diluting liquid chamber.

[0008] As a preferred embodiment of the present invention, the valve is a hot-melt material layer, and the hot-melt material in the hot-melt material layer is wax, thermosensitive gel or thermoplastic polymers such as polyolefin, polyester and the like.

[0009] The second object of the present invention is to provide a microfluidic chip, which includes a chip body and the controlled release reagent storage box as described above; the chip body is provided with a accommodating cavity adapted to the controlled release reagent storage box, and the controlled release reagent storage box is arranged in the accommodating cavity.

[0010] As a preferred embodiment of the present invention, the chip body comprises a connected sample lysis pool, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative subpackaging area and a reaction detection area, wherein the nucleic acid extraction area and the quantitative subpackaging area are respectively connected to a waste liquid tank through a microfluidic channel; the nucleic acid dilution area, the quantitative subpackaging area and the waste liquid tank are respectively connected to the atmosphere through a microfluidic channel, and the reaction detection area has a transparent area for fluorescence detection; The accommodating chamber is divided into a first accommodating chamber and a second accommodating chamber by a partition rib. The bottom wall of the first accommodating chamber protrudes upward and is provided with a plurality of positioning support parts for positioning and supporting the controlled-release reagent storage box. The sample lysis pool is formed between the bottom wall of the first accommodating chamber and the top surface of the positioning support part; the nucleic acid dilution area includes a diluent release pool, and the diluent release pool is connected to the second accommodating chamber.

[0011] As a preferred embodiment of the present invention, the nucleic acid extraction area includes a mixing buffer pool and a nucleic acid extraction column; the mixing buffer pool is respectively connected to the sample lysis pool and the nucleic acid extraction column, and the nucleic acid extraction column is respectively connected to the nucleic acid dilution area and the waste liquid tank through a microchannel; the nucleic acid extraction column includes an extraction cavity with a cylindrical structure and a pressure ring sealed and installed in the extraction cavity, a small hole for liquid outflow is opened on the bottom wall of the extraction cavity, an extraction membrane is arranged on the bottom wall of the extraction cavity, the pressure ring is against the extraction membrane, and is used to limit the position of the extraction membrane in the flow direction of the liquid.

[0012] As a preferred embodiment of the present invention, a paraffin valve is provided on the microchannel between the nucleic acid extraction column and the waste liquid tank.

[0013] As a preferred embodiment of the present invention, the nucleic acid dilution area also includes a dilution pool, which is connected to the dilution liquid release pool through a zigzag microfluidic channel. The dilution pool is connected to the nucleic acid extraction area and the quantitative filling area through microfluidic channels, respectively. The microfluidic channel between the dilution pool and the quantitative filling area is an S-shaped or inverted U-shaped microfluidic channel.

[0014] As a preferred embodiment of the present invention, the quantitative filling area includes a liquid channel and a plurality of quantitative grooves arranged at intervals, the liquid channel is connected to the nucleic acid dilution area and the waste liquid groove through microfluidics, and each of the quantitative grooves is connected to the liquid channel respectively; a downwardly recessed rectifier pool is provided at the connection between the liquid channel and the microfluidic channel connected to the nucleic acid dilution area.

[0015] As a preferred embodiment of the present invention, the reaction detection area includes a plurality of reaction chambers arranged in one-to-one correspondence with the quantitative grooves, and the reaction chambers are connected to the quantitative grooves through microchannels; the reaction chambers are pre-installed with LAMP reagent freeze-dried microspheres and fluorescent probes; a paraffin groove is arranged above the reaction chamber, and the paraffin groove is filled with paraffin; a blocking groove connected to the paraffin groove is arranged on the microchannel between the reaction chamber and the quantitative groove.

[0016] As a preferred embodiment of the present invention, the microfluidic chip further comprises a covering film, and the covering film covers the chip body; the covering film is transparent and is preferably a pressure-sensitive film or a heat-sensitive film.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The controlled-release reagent storage box of the present invention integrates the pre-storage of multiple reagents through the cooperation of the sealing film and the box body, and a valve for controlling the release of the reagent is provided in the release hole of each reagent storage chamber, which can well meet the independent release requirements of multiple reagents and multi-step reactions, and at the same time can ensure the safety and stability of the reagents during long-term storage and the flexible release in sequence during the reaction process, effectively preventing the leakage of reagents; compared with the traditional manual addition of reagents, the operation process is simplified, the human error is greatly reduced, and the reliability of the experiment is improved. At the same time, by providing a sample addition port on the controlled-release reagent storage box, it can be adapted for use with a microfluidic chip. Compared with the existing technology of directly storing the reagents in the chip, the sealing is better, and the problems of reagent volatilization, leakage and cross contamination are effectively avoided, and the reliability and repeatability of the experimental results are improved to a certain extent, and it is particularly suitable for biological experiments with high requirements for reagent stability.

[0018] The microfluidic chip of the present invention comprises a chip body and the above-mentioned controlled-release reagent storage box. By installing the controlled-release reagent storage box in the accommodating cavity of the chip body, it can not only well meet the complex experimental process of multiple reagents and multiple steps, but also take into account portability, integration and automation while simplifying the structure of the chip body, thereby further reducing the manufacturing cost of the chip, meeting the needs of fast and accurate experiments, and facilitating the promotion and application of microfluidic chips in complex biological molecule analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a three-dimensional diagram of the controlled-release reagent storage box of the present invention; Figure 2 A three-dimensional diagram of the controlled-release reagent storage box of the present invention from another angle; Figure 3 This is a front view of the controlled release reagent storage box of the present invention; Figure 4 A bottom view of the controlled-release reagent storage box of the present invention; Figure 5 is a three-dimensional diagram of the microfluidic chip of the present invention; Figure 6 This is a front view of the microfluidic chip of the present invention; Figure 7 A three-dimensional diagram of the chip body of the present invention; Figure 8 It is a front view of the chip body of the present invention; Fig. 9 For the present invention Figure 8 BB section view; Description of the accompanying drawings: 100, box body; 110, reagent storage chamber; 111, first cleaning liquid chamber; 112, elution liquid chamber; 113, second cleaning liquid chamber; 114, diluent chamber; 120, release hole; 130, bump; 140, sample injection port; 150, avoidance groove; 200, chip body; 210, receiving chamber; 211, partition rib; 212, first receiving chamber; 213, second receiving chamber; 214, positioning support; 215, sample lysis pool; 220, nucleic acid extraction area; 22 1. Mixing buffer tank; 222. Nucleic acid extraction column; 2221. Extraction chamber; 2222. Pressure ring; 2223. Small hole; 2224. Extraction membrane; 223. Guide groove; 230. Nucleic acid dilution area; 231. Dilution liquid release tank; 232. Dilution tank; 240. Quantitative filling area; 241. Liquid channel; 242. Quantitative tank; 243. Rectifier tank; 250. Reaction detection area; 251. Reaction chamber; 252. Paraffin tank; 253. Blocking tank; 260. Waste liquid tank; 270. Paraffin valve. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] like Figure 1~Figure 4 As shown, the reagent storage box for centrifugal fluidics of the present invention includes a box body 100 and a sealing film (not shown) covering the box body 100. The sealing film can effectively physically isolate the reagents in the box body 100, improve the sealing of the reagents, and ensure the safety and anti-leakage performance of long-term storage. Preferably, the sealing film is an aluminum film covered on the box body 100 by heat sealing. Specifically, the top of the box body 100 is recessed downward to open a plurality of mutually independent reagent storage chambers 110, and each reagent storage chamber 110 is provided with a release hole 120 at a position close to the bottom wall of the side wall, and an adjustable valve for controlling the release of the reagent is provided in the release hole 120 to close the reagent storage chamber 110. The side wall of the box body 100 is extended outward to be provided with a protrusion 130, and the protrusion 130 is provided with a through sample injection port 140, and the protrusion 130 plays a positioning role. It can be seen that the reagent storage box of the present invention can integrate the pre-storage of multiple reagents into one, and by adjusting the valve in the release hole 120 of each reagent storage cavity 110, the multiple reagents can be released independently in sequence, which can well meet the independent release requirements of multiple reagents and multi-step reactions, and at the same time ensure the safety and stability of the reagents during long-term storage and the flexible release on demand during the reaction process, effectively preventing the leakage of reagents. At the same time, by providing a sample addition port 140 on the controlled release reagent storage box, it can be adapted for use with a microfluidic chip. Compared with the prior art of directly storing reagents in the chip, the sealing is better, which improves the reliability of the experimental results to a certain extent.

[0022] In some embodiments, Figure 3 As shown, the multiple reagent storage chambers 110 are respectively a first cleaning liquid chamber 111, an eluting liquid chamber 112, a second cleaning liquid chamber 113 and a diluting liquid chamber 114. Among them, the eluting liquid chamber 112 is arranged between the first cleaning liquid chamber 111 and the second cleaning liquid chamber 113, and the second cleaning liquid chamber 113 is arranged between the eluting liquid chamber 112 and the diluting liquid chamber 114. In order to enable the reagent storage box to be compatible with the microfluidic chip, an avoidance groove 150 is provided on the bottom surface of the box body 100 corresponding to the position between the second cleaning liquid chamber 113 and the diluting liquid chamber 114. Of course, the reagent storage chamber 110 of the present invention is not limited to the above-mentioned structural setting, and the specific structural setting of the reagent storage chamber 110 can be adjusted according to the different uses of the microfluidic chip.

[0023] In some embodiments, the valve (not shown) in the release hole 120 is a hot melt material layer, and the hot melt material in the hot melt material layer is one of wax, thermosensitive gel or thermoplastic polymer (such as polyolefin, polyester, etc.). In actual use, the reagent storage cavity 110 can be opened by melting the hot melt material layer to release the reagent; wherein, the hot melting method preferably adopts laser hot melting, and laser hot melting can be targeted to perform laser irradiation on the hot melt material layer in a certain release hole 120, and the laser intensity and irradiation time can be adjusted according to the reagent needs, effectively ensuring the independent, sequential and precise release of multiple reagents to meet the complex needs of multi-step reactions, and suitable for reactions that are sensitive to heating. In some embodiments, the hot melt material layer can also be a low melting point polymer, and the low melting point polymer can also be hot melted by heating with a laser or an electric heating element, which can also achieve independent control of the release order and time of each reagent, and at the same time can increase the temperature resistance or stability of reagent storage and. Of course, the valve can also be a micro-mechanical valve such as a solenoid valve or a micro-switch valve, which uses an electrical signal to control the opening and closing of the valve, so as to achieve the effect of independent release of each reagent in sequence, which is suitable for multi-step biochemical reaction requirements. In some embodiments, the valve is a pH-sensitive material layer, such as a chitosan-based composite material, a pH-sensitive hydrogel, etc., and the material is immediately degraded by introducing an acid-base regulator during use to release the reagent. This method does not require the addition of mechanical devices or heat source equipment such as lasers, and is suitable for specific experimental requirements. In some embodiments, the valve is a micro-pressure valve, which automatically opens the pressure valve to release the reagent when the centrifugal force reaches the threshold. This method uses centrifugal force and pressure changes to control the release of the reagent, reduces the dependence on external equipment, and can meet the needs of multi-step reactions. In some embodiments, the valve is a diaphragm with a certain thickness, and mechanical pressure or vibration is used to rupture the diaphragm to achieve the release of the reagent. The layer structure design of the diaphragm is used to achieve the sequential release of different reagents, which can also meet the needs of multi-step experiments, while avoiding the use of heat sources, and is suitable for the storage of thermosensitive reagents. In some embodiments, the valve is an adjustable microporous membrane, which adjusts the opening and closing degree of the micropores by changing external environmental factors such as temperature, humidity or electric field, thereby achieving controlled and sequential release of multiple reagents, avoiding the use of mechanical and thermal devices, and is suitable for specific automated biochemical experimental scenarios.

[0024] In summary, the controlled-release reagent storage box of the present invention can store multiple reagents, and the release of each reagent can be independently controlled, with good sealing performance, which effectively ensures the stability of the reagent during long-term storage and the flexible release during the reaction process.

[0025] like Figure 5 and Figure 6As shown, the present invention also provides a microfluidic chip, which includes a covering film, a chip body 200 and the above-mentioned controlled release reagent storage box; the chip body 200 is provided with a receiving cavity 210 adapted to the controlled release reagent storage box, the controlled release reagent storage box is arranged in the receiving cavity 210, and the covering film covers other positions of the chip body 200 except the receiving cavity 210. The microfluidic chip integrates multiple reagents into the controlled release reagent storage box, takes into account portability, integration and automation, and simplifies the structure of the chip body 200, thereby further reducing the manufacturing cost of the chip, can well meet the complex experimental process of multiple reagents and multiple steps, meet the requirements of fast and accurate experiments, and is conducive to the promotion and application of microfluidic chips in complex biological molecule analysis.

[0026] Specifically, Figure 7 and Figure 8As shown, the chip body 200 is a fan-shaped structure, and the accommodating chamber 210 is arranged at a position close to the center of the chip body 200. Specifically, the accommodating chamber 210 is divided into a first accommodating chamber 212 and a second accommodating chamber 213 by a partition rib 211. The bottom wall of the first accommodating chamber 212 is protruding upward and is provided with a plurality of positioning support parts 214 for positioning and supporting the controlled release reagent storage box. The space between the bottom wall of the first accommodating chamber 212 and the top surface of the positioning support part 214 forms a sample lysis pool. The sample addition port 140, the first cleaning liquid chamber 111, the eluting liquid chamber 112 and the second cleaning liquid chamber 113 in the controlled release reagent storage box are arranged in the first accommodating chamber 212, and the diluting liquid chamber 114 is arranged in the second accommodating chamber 213. The chip body 200 also includes a nucleic acid extraction area 220, a nucleic acid dilution area 230, a quantitative subpackaging area 240 and a reaction detection area 250 which are sequentially distributed along the radial direction of the chip body 200. The sample lysis pool, the nucleic acid extraction area 220, the nucleic acid dilution area 230, the quantitative subpackaging area 240 and the reaction monitoring area are sequentially connected. The nucleic acid extraction area 220 and the quantitative subpackaging area 240 are respectively connected to the waste liquid tank 260 through microchannels; the nucleic acid dilution area 230, the quantitative subpackaging area 240 and the waste liquid tank 260 are respectively connected to the atmosphere through microchannels, and the reaction detection area 250 has a transparent area for fluorescence detection, and the position corresponding to the transparent area on the cover plate is transparent. The nucleic acid dilution area 230 includes a diluent release pool 231, and the diluent release pool 231 is connected to the second accommodating chamber 213. The sample loading port 140 of the controlled release reagent storage box is located above the sample lysis pool 215 and is connected to the sample lysis pool 215. The sample and the lysis solution are injected into the sample lysis pool through the sample loading port 140 and mixed under the action of centrifugal force. The external heating plate provides a uniform heat source to the upper and lower surfaces of the microfluidic chip for heating and lysis, thereby realizing the release of nucleic acid. The lysed sample enters the nucleic acid extraction area 220 under the action of centrifugal force. The controlled-release reagent storage box is installed in the accommodating cavity 210 so that the release hole 120 of each reagent storage cavity 110 is located in the nucleic acid extraction area 220. Under the action of the laser, the valve in the release hole 120 opens to release the first cleaning liquid, the second cleaning liquid and the elution liquid in sequence to clean, capture and elute the sample. The eluted nucleic acid is diluted in the nucleic acid dilution area 230, and then flows through the quantitative filling area 240 to be quantitatively filled into the reaction detection area 250 for amplification reaction and fluorescence detection. The liquid in the nucleic acid extraction area 220 and the excess sample liquid are drained into the waste liquid tank 260 through the microchannel for collection.It can be seen that the microfluidic chip of the present invention integrates sample lysis, nucleic acid extraction and dilution, quantitative packaging, amplification reaction and fluorescence detection on the chip body 200 through a modular design, and connects each area through a microchannel, relies on centrifugal force to drive the flow of liquid, and has highly integrated functions, thereby realizing the full process integration, automation and precise control of fluids for nucleic acid detection. It is easy to operate and can complete the detection process without external auxiliary equipment. It greatly improves the detection efficiency and the accuracy of the test results while reducing the equipment procurement and maintenance costs, and also greatly reduces the difficulty of operation, which can well meet the needs of high-throughput and rapid detection.

[0027] Specifically, the nucleic acid extraction area 220 includes a mixing buffer pool 221 and a nucleic acid extraction column 222 arranged in sequence from top to bottom, thereby ensuring that the flow path of the reagent under the action of centrifugal force is a unidirectional flow to avoid cross contamination. The sample lysis pool 215 is directly connected to the mixing buffer pool 221, and the mixing buffer pool 221 is connected to the nucleic acid extraction column 222. The nucleic acid extraction column 222 is connected to the nucleic acid dilution area 230 and the waste liquid tank 260 through microchannels. More specifically, as Fig. 9As shown, the nucleic acid extraction column 222 includes an extraction chamber 2221 with a cylindrical structure and a pressure ring 2222 which is sealed and installed in the extraction chamber 2221 and has an interference fit with the extraction chamber 2221. The extraction chamber 2221 is integrally formed in the chip body 200. A small hole 2223 which is connected to the extraction chamber 2221 is provided on the bottom wall of the extraction chamber 2221. The inner diameter of the small hole 2223 is smaller than the inner diameter of the extraction chamber 2221, so as to allow liquid to flow out. An extraction membrane 2224 is provided on the bottom wall of the extraction chamber 2221. The extraction membrane 2224 is provided at the connection between the small hole 2223 and the extraction chamber 2221. The extraction membrane 2224 is preferably a silicone mold. The pressure ring 2222 abuts against the extraction membrane 2224 and is used to limit the position of the extraction membrane 2224 in the flow direction of the liquid, so as to ensure that the extraction membrane 2224 is stable and the fluid flow path is controlled. The sample liquid enters the nucleic acid extraction column 222 through the mixing buffer tank 221 by centrifugal force. The first cleaning liquid and the second cleaning liquid are released by laser thermal melting and flow through the nucleic acid extraction column 222 in sequence under the action of centrifugal force into the waste liquid tank 260 to achieve graded cleaning, which can effectively remove impurities in the sample liquid. The extraction membrane 2224 can efficiently adsorb nucleic acids. After the cleaning is completed, the eluent is released by laser thermal melting and flows through the extraction column under the action of centrifugal force to elute the purified nucleic acid into the downstream nucleic acid dilution area 230. Furthermore, the microchannel between the nucleic acid extraction column 222 and the waste liquid tank 260 and the nucleic acid dilution area 230 is an inverted Y-shaped shunt structure, and the liquid flow direction can be controlled by controlling the centrifugal direction. Specifically, the cleaning liquid enters the waste liquid tank 260 by making the microfluidic chip rotate counterclockwise, and the eluted nucleic acid enters the nucleic acid dilution area 230 by making the microfluidic chip rotate clockwise. The present invention embeds the nucleic acid extraction column 222 into the microfluidic chip and combines it with the microfluidic channel, utilizes centrifugal force to realize the automatic flow and separation of liquid, avoids sample loss and external interference, ensures high extraction efficiency and stable nucleic acid purity. In order to ensure that the liquid in the extraction chamber 2221 can be discharged quickly, the inner diameter of the small hole 2223 cannot be too small, which makes it difficult for the inner diameter of the small hole 2223 to match the width of the microchannel. Therefore, a guide groove 223 is arranged below the accommodating chamber, and the bottom wall of the guide groove 223 is an inclined surface, which can better play the role of confluence and diversion. Of course, the nucleic acid extraction column 222 can also be installed in the chip body 200 as an independent component by assembly, so that the extraction membrane 2224 and the pressure ring 2222 are easy to install.

[0028] Furthermore, a paraffin valve 270 is provided on the microchannel between the nucleic acid extraction column 222 and the waste liquid tank 260, and the outlet of the paraffin valve 270 is located below the nucleic acid dilution zone 230, and solid paraffin is filled in the paraffin valve 270. During the sample cracking and graded cleaning process, the microfluidic chip is centrifuged counterclockwise to allow the waste liquid to enter the waste liquid tank 260 through the microchannel. After the above process is completed, the paraffin in the paraffin valve 270 is heated to melt it and enter the microchannel below. The paraffin in the microchannel is cooled and solidified by cooling, thereby closing the microchannel connected to the waste liquid tank 260, and then the eluent is collected by centrifugation clockwise to enter the nucleic acid dilution zone 230, which can not only improve the yield of nucleic acid and avoid waste, but also well avoid the waste liquid in the waste liquid tank 260 from contaminating the eluted nucleic acid, and to a certain extent ensure the purity of the nucleic acid.

[0029] Specifically, the nucleic acid dilution area 230 also includes a dilution pool 232, which is connected to the dilution liquid release pool 231 through a zigzag microchannel, and the dilution pool 232 is connected to the atmosphere through a microchannel. The dilution pool 232 is arranged on one side of the mixing buffer pool 221 and is slightly lower than the mixing buffer pool 221. The dilution pool 232 is connected to the nucleic acid extraction area 220 and the quantitative subpackaging area 240 through microchannels, and the microchannel between the dilution pool 232 and the quantitative subpackaging area 240 is an S-shaped or inverted U-shaped microchannel. After the dilution liquid in the dilution liquid chamber 114 is released by laser thermal melting, it enters the dilution pool 232 under the action of centrifugal force to dilute the eluted nucleic acid, and the diluted nucleic acid sample liquid to be tested enters the quantitative subpackaging area 240 under the drive of centrifugal force.

[0030] Specifically, the quantitative subpackaging area 240 includes a liquid channel 241 and a plurality of equal-volume quantitative grooves 242 arranged at intervals. The liquid channel 241 is connected to the nucleic acid dilution area 230 and the waste liquid tank 260 through microchannels, respectively. Each quantitative groove 242 is connected to the liquid channel 241, and the nucleic acid sample liquid to be tested is driven into the plurality of equal-volume quantitative grooves 242 by centrifugal force, so as to achieve accurate quantitative subpackaging of the sample and ensure the quantitative consistency of the sample amplification reaction, effectively ensure the sensitivity, accuracy and reliability of the subsequent amplification reaction, and well avoid the problem of quantitative errors caused by manual operation or complex equipment in the prior art, thereby affecting the amplification results. In order to further ensure the quantitative accuracy of sample subpackaging, a downwardly concave rectifying pool 243 is provided at the connection between the liquid channel 241 and the microchannel connected to the nucleic acid dilution area 230. The rectifying pool 243 can play a buffering role for the sample liquid entering the quantitative subpackaging area 240, so as to avoid the problem of quantitative inaccuracy caused by the flow rate and flow deviation of the sample liquid in the first reaction chamber 251.

[0031] Specifically, Figure 6As shown, the reaction detection area 250 includes a plurality of reaction chambers 251 arranged in one-to-one correspondence with the quantitative grooves 242. The reaction chambers 251 are connected to the quantitative grooves 242 through microchannels, and the sample enters the reaction chamber 251 from the quantitative grooves 242. Each reaction chamber 251 is pre-installed with LAMP reagent (loop-mediated isothermal amplification reagent) freeze-dried microspheres and fluorescent probes. After the sample enters the reaction chamber 251, the LAMP reagent freeze-dried microspheres can be quickly and automatically re-dissolved to ensure the reaction sensitivity and specificity. A paraffin tank 252 is arranged above the reaction chamber 251, and the paraffin tank 252 is filled with paraffin. A blocking tank 253 connected to the paraffin tank 252 is arranged on the microchannel between the reaction chamber 251 and the quantitative groove 242. The present invention adopts paraffin wax plugging technology. After the paraffin wax is melted by heating before the amplification reaction, the microchannel at the entrance of the reaction chamber 251 is plugged by centrifugal force to form a sealing layer, which physically isolates the aerosol pollution source, can effectively avoid aerosol leakage and cross contamination between samples during the amplification process, thereby reducing the risk of false positive results, significantly improving the detection specificity, safety and accuracy, and is particularly suitable for multi-sample high-throughput detection scenarios. Of course, the plugging agent is not limited to paraffin, and other commonly used plugging agents in the field can also be used. In order to improve the sealing of paraffin to the entrance of the reaction chamber 251, the plugging groove 253 is preferably in a V-shaped structure, and the microchannel between the plugging groove 253 and the paraffin groove 252 is tilted, so that the paraffin can achieve rapid sealing of the plugging groove 253 under the action of centrifugal force. Further, the bottom wall of the reaction chamber 251 is transparently set, and accordingly, the position of the cover layer corresponding to the reaction chamber 251 is also transparently set to form a transparent detection window. After the amplification reaction is completed, an external fluorescence detection module is used to collect real-time fluorescence signal data through a transparent detection window, and the data is analyzed and displayed on an external Android all-in-one device.

[0032] In summary, the microfluidic chip of the present invention integrates and stores multiple reagents in a controlled-release reagent storage box, and utilizes the valve in the release hole 120 to realize the timed sequential release of multiple reagents, thereby effectively improving integration and automation. At the same time, it simplifies the structure of the microfluidic chip, reduces its manufacturing cost, and improves the universality of the microfluidic chip. It only needs to replace the controlled-release reagent storage box storing different types of reagents to be applicable to different biological molecule analysis experiments such as nucleic acid detection and protein molecules.

[0033] Taking nucleic acid detection as an example, the method for using the above-mentioned microfluidic chip specifically includes the following steps: S1, install the controlled release reagent storage box in the receiving cavity 210 of the chip body 200, pre-place paraffin in the paraffin valve 270, and pre-place paraffin, LAMP reagent freeze-dried microspheres and fluorescent probes in the reaction detection area 250; S2, adding the sample and the lysate into the sample lysing pool 215 through the sample adding port 140 and mixing them evenly, heating (e.g., heating at 56° C. for 10 min) to lyse the sample to release the nucleic acid, and obtaining a lysate mixture after the lysate is complete; centrifuging the microfluidic chip to allow the lysate mixture to enter the nucleic acid extraction column 222 in the nucleic acid extraction area 220 and then enter the waste liquid tank 260; S3, the valve in the release hole 120 is opened by laser irradiation, the first cleaning liquid is released, the microfluidic chip is centrifuged counterclockwise, the first cleaning liquid passes through the nucleic acid extraction column 222 to perform primary cleaning on the sample and then enters the waste liquid tank 260; the valve in the release hole 120 is opened by laser irradiation, the second cleaning liquid is released, the microfluidic chip is centrifuged, the second cleaning liquid passes through the nucleic acid extraction column 222 to perform secondary cleaning on the sample and then enters the waste liquid tank 260; S4, opening the valve in the release hole 120 by laser irradiation to release the eluent, rotating the microfluidic chip clockwise to allow the eluent to elute the captured nucleic acid through the nucleic acid extraction column 222, and allowing the eluted nucleic acid to enter the nucleic acid dilution area 230; S5, opening the valve in the release hole 120 by laser irradiation to release the diluent, centrifuging the microfluidic chip to mix the nucleic acid and the diluent in the nucleic acid dilution area 230 and dilute them to an ideal concentration for subsequent amplification; S6. The diluted nucleic acid sample solution to be tested enters the quantitative dispensing area 240 driven by centrifugal force, so that the nucleic acid sample solution to be tested is quantitatively dispensed into the reaction detection area 250; S7, the nucleic acid sample liquid to be tested enters the reaction detection area 250 and re-dissolves the LAMP reagent freeze-dried microspheres, heats the paraffin to melt, and the molten paraffin blocks the microchannel between the reaction detection area 250 and the quantitative filling area 240 under the action of centrifugal force; the re-dissolved and mixed liquid is heated to perform quantitative amplification reaction and fluorescence signal detection.

[0034] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A controlled release reagent storage box for centrifugal fluidics, characterized in that: It comprises a box body and a sealing film covering the box body; the top of the box body is recessed downward to form a plurality of mutually independent reagent storage chambers, each of the reagent storage chambers is provided with a release hole on the side wall near the bottom wall, and a valve for controlling the release of the reagent is provided in the release hole; a convex block is extended outwardly from one side of the box body, and a sample addition port is provided on the convex block.

2. The controlled release reagent storage box for centrifugal fluidics according to claim 1, characterized in that: The multiple reagent storage chambers are respectively a first cleaning liquid chamber, an elution liquid chamber, a second cleaning liquid chamber and a dilution liquid chamber; The elution liquid chamber is arranged between the first cleaning liquid chamber and the second cleaning liquid chamber, and the second cleaning liquid chamber is arranged between the elution liquid chamber and the dilution liquid chamber.

3. The controlled release reagent storage box for centrifugal fluidics according to claim 2, characterized in that: An avoidance groove is provided on the bottom surface of the box body at a position corresponding to between the second cleaning liquid chamber and the diluting liquid chamber.

4. The controlled-release reagent storage box for centrifugal fluidics according to claim 1 or 2, characterized in that: The valve is a hot-melt material layer, and the hot-melt material in the hot-melt material layer is wax, thermosensitive gel or thermoplastic polymer.

5. A microfluidic chip, characterized in that: It comprises a chip body and a controlled-release reagent storage box as described in any one of claims 1 to 4; the chip body is provided with a receiving cavity adapted to the controlled-release reagent storage box, and the controlled-release reagent storage box is arranged in the receiving cavity.

6. The microfluidic chip according to claim 5, characterized in that: The chip body comprises a connected sample lysis pool, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative subpackaging area and a reaction detection area, wherein the nucleic acid extraction area and the quantitative subpackaging area are respectively connected to a waste liquid tank through a microfluidic channel; the nucleic acid dilution area, the quantitative subpackaging area and the waste liquid tank are respectively connected to the atmosphere through a microfluidic channel, and the reaction detection area has a transparent area for fluorescence detection; The accommodating chamber is divided into a first accommodating chamber and a second accommodating chamber by a partition rib. The bottom wall of the first accommodating chamber protrudes upward and is provided with a plurality of positioning support parts for positioning and supporting the controlled-release reagent storage box. The sample lysis pool is formed between the bottom wall of the first accommodating chamber and the top surface of the positioning support part; the nucleic acid dilution area includes a diluent release pool, and the diluent release pool is connected to the second accommodating chamber.

7. The microfluidic chip according to claim 6, characterized in that: The nucleic acid extraction area includes a mixing buffer pool and a nucleic acid extraction column; the mixing buffer pool is connected to the sample lysis pool and the nucleic acid extraction column respectively, and the nucleic acid extraction column is connected to the nucleic acid dilution area and the waste liquid tank respectively through a microchannel; the nucleic acid extraction column includes an extraction cavity with a cylindrical structure and a pressure ring sealed and installed in the extraction cavity, a small hole for liquid outflow is opened on the bottom wall of the extraction cavity, an extraction membrane is arranged on the bottom wall of the extraction cavity, the pressure ring is against the extraction membrane, and is used to limit the position of the extraction membrane in the flow direction of the liquid.

8. The microfluidic chip according to claim 7, characterized in that: A paraffin valve is arranged on the microchannel between the nucleic acid extraction column and the waste liquid tank.

9. The microfluidic chip according to claim 6, characterized in that: The quantitative filling area includes a liquid channel and a plurality of quantitative grooves arranged at intervals. The liquid channel is connected to the nucleic acid dilution area and the waste liquid groove through microfluidics respectively, and each of the quantitative grooves is connected to the liquid channel respectively; a downwardly recessed rectifying pool is provided at the connection between the liquid channel and the microfluidic channel connected to the nucleic acid dilution area.

10. The microfluidic chip according to claim 6, characterized in that: The reaction detection area includes a plurality of reaction chambers arranged in one-to-one correspondence with the quantitative grooves, and the reaction chambers are connected to the quantitative grooves through microchannels; the reaction chambers are pre-installed with LAMP reagent freeze-dried microspheres and fluorescent probes; a paraffin groove is arranged above the reaction chamber, and the paraffin groove is filled with paraffin; a blocking groove connected to the paraffin groove is arranged on the microchannel between the reaction chamber and the quantitative groove.

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